Витамин D Дополняет медиацию, переходя к борьбе с воспламенением Цитокина при множественном склерозе.

Highlights of the Study

High levels of pro-inflammatory cytokines were found in drug-naïve MS patients.

Above finding was accompanied by a low level of serum vitamin D (Vit-D).

Vit-D supplementation brought a significant decrease in pro-inflammatory cytokines.

The ratios of several pro-inflammatory to anti-inflammatory cytokines declined after Vit-D supplementation.

Vit-D supplementation may have a protective role in MS and is of therapeutic importance.

Introduction

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and demyelination within the central nervous system (CNS) [ 1 ]. This pathological process involves dysregulation of the immune response, with increased production of pro-inflammatory cytokines such as interleukin (IL)-2, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), along with reduced levels of anti-inflammatory cytokines like transforming growth factor-beta (TGF-β) and IL-13 [ 1 , 2 ].

The vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily, is expressed on immune cells, including activated T lymphocytes, dendritic cells, antigen-presenting cells, and mononuclear cells [ 3 ]. Previous research suggests a connection between vitamin D (Vit-D) status and autoimmune diseases [ 3 – 5 ]. Significant levels of VDR in immune cells, combined with the ability of activated dendritic cells to produce the active Vit-D hormone, imply that Vit-D may have immunoregulatory properties [ 3 , 4 ].

Epidemiological studies support the protective role of Vit-D in autoimmune diseases. For instance, a large population study reported a 40% lower risk of MS in women with the highest quintile of Vit-D intake [ 6 ]. Similarly, data from the Women’s Iowa Health Study, involving 29,368 women, revealed an inverse relationship between Vit-D intake and rheumatoid arthritis risk [ 7 ]. In animal models of MS, Vit-D deficiency exacerbates experimental autoimmune encephalomyelitis (EAE), while Vit-D supplementation inhibits its recurrence [ 8 ]. An Australian study further suggests that Vit-D and sun exposure may help prevent MS by promoting T-regulatory cell activation and IL-10 release [ 9 ], which suppresses TH17-mediated inflammation [ 10 ]. While Vit-D supplementation has been shown to increase serum levels of IL-10 and TGF-β1 in MS patients, indicating anti-inflammatory effects, its impact on cytokine regulation and disability outcomes remains controversial [ 11 ].

Given the critical role of cytokines in mediating autoimmune and inflammatory tissue damage, this study aimed to evaluate the impact of Vit-D supplementation on cytokine profiles in a cohort of drug-naïve, newly diagnosed MS patients; this research would contribute to a better understanding of the immunomodulatory effects of Vit-D in MS.

Methods

Subjects

In this observational study, we included 16 newly diagnosed drug-naïve adult Kuwaiti MS patients belonging to the age group of 20–40 years. The necessary sample size (at least 10 patients) was manually determined considering that MS is an uncommon condition in Kuwait, where incidence is reported at about 2.62/100,000 [ 12 ]. Kuwait is a small country and is home to only 1,546,108 Kuwaiti nationals. According to the most recent census data, 381,932 Kuwaiti citizens belong to the 20–40 age group, [ 13 ]. The subjects presented with MS symptoms for the very first time and visited the specialized MS clinic at Mubarak Al-Kabeer Hospital, Kuwait. All the patients were carefully examined by a team of experienced neurologists, and the MS diagnosis was confirmed according to the McDonald criteria [ 14 ]. Following diagnosis, subjects were recruited into the study. This study complied with the Declaration of Helsinki. Informed voluntary consent was obtained from all participants, which was approved by the Ethics Committees of the Health Sciences Centre, Kuwait University, Kuwait, Grant No. MM 03/09.

At the time of recruitment, none of the subjects were on disease-modifying therapy or any prescribed medications. Patients were monitored at 6-month intervals and followed for 12 months. Information was collected on demographic parameters, including age, gender, and BMI. Moreover, clinical parameters like presenting symptoms, apparent clinical phenotype, severity of disease, and past medical history including over-the-counter dietary supplements were noted at recruitment. The clinical course of the disease was classified as relapsing-remitting, primary progressive, secondary progressive, and progressive relapsing [ 15 ]. Disabilities were stratified according to the expanded disability status scale (EDSS) (16). The EDSS rates the clinical status of a patient on a 0–10 scale, where a score of 0 indicates that the patient is normal, whereas a score of 10 denotes death. Benign MS was defined as an EDSS of ≤3. A relapse was defined as the emergence of new or previous signs or symptoms that lasted for more than 24 h [ 16 ].

Biochemical and Immunological Analysis

All subjects provided overnight fasted blood specimens for liver function and kidney function tests, including serum levels of intact parathyroid hormone (iPTH) (measured by routine auto-analyzer techniques [Hitachi 911 and Beckman LX-20] following manufacturer instructions). The intra-assay coefficients of variations (CVs) of iPTH assays were 2.4% and 2.3% at 0.83 and 9.4 pmol/L, respectively. The inter-assay CVs were 3.3% and 2.3% at 1.3 and 9.4 nmol/L, respectively. The total 25-hydroxy Vit-D was measured by an enzyme immunoassay using the 25-hydroxy Vit-D enzyme immunoassay kit, Immunodiagnostic Systems. The total 25-hydroxy Vit-D2+D3 [25(OH)D] level was categorized as <50 = deficient, 50–74 = insufficient, 75–250 = sufficient (unit expressed as nmol/L), according to the manufacturer’s instructions. The procedure for estimating 25(OH)D in serum involves dilution of calibrators, controls, and samples with biotin labeled 25(OH)D. The diluted samples were incubated in highly specific sheep 25(OH)D antibody-coated microtiter wells for 2 h at room temperature before aspiration and washing. Enzyme (horseradish peroxidase), labeled avidin, was added and binds selectively to complexed biotin, and following a further wash step, color was developed using a chromogenic substrate (TMB). The absorbance of the stopped reaction mixtures was read in a microtiter plate reader. The color intensity developed was proportional to the concentration of 25(OH)D. The intra-assay CVs were 5.3% and 5.6% at 39.0 and 61.1 nmol/L, respectively. The inter-assay CVs were 4.6% and 6.4% at 40.3 and 72.0 nmol/L, respectively.

For the estimation of cytokine production, the following procedure was used: peripheral blood mononuclear cells (PBMC) were isolated from venous blood by density gradient centrifugation on Ficoll Hypaque (Pharmacia Biotech, Uppsala, Sweden) and then aliquoted into 96-well tissue culture plate wells at a density of 10 5 cells per well. PBMC were stimulated with the mitogen phytohemagglutinin (PHA) (Sigma Chemicals, USA) at a concentration of 5 µg/mL for 96 h. PBMC were also cultured with RPMI alone as a control. The proliferation of PBMC was assessed by radiolabeled thymidine uptake. Culture supernatants were collected, centrifuged for 5 min at 5,000 g , and then stored at −80°C to estimate cytokine levels. We measured levels of the inflammatory Th1 cytokines IFN-γ and TNF-α, the Th1-inducing cytokine IL-12, and the anti-inflammatory Th2 cytokines IL-4, IL-5, and IL-10 by ELISA (Human Interleukin ELISA kits, eBioscience ThermoFisher Scientific, USA). All the assays were performed according to the manufacturer’s protocols.

Follow-Up Studies

After analysis of the fasting blood samples collected at recruitment, the patients were given Vit-D supplementation, depending on their intact parathyroid hormone (iPTH) level. Patients with a high iPTH value (>9.3 pmol/L) were treated with oral Vit-D3 (50,000 IU per week, along with Caltrate D twice a day), while those with iPTH level within the identified specific reference range (1.3–9.3 pmol/L for the Kuwaiti population) were treated with oral Caltrate D twice a day. The rationale for stratifying patients into low-versus high-dose Vit-D based on iPTH levels stemmed from concerns about the potential risk of hypervitaminosis D, particularly if high doses (e.g., 50,000 units per week) were administered to patients with normal iPTH levels. Patients were also prescribed IFN-β1α therapy. All clinical, radiologic, and laboratory parameters (as mentioned above) were monitored at 6-month intervals until the end of the 12-month follow-up. During the follow-up, we measured disease activities through both clinical and radiological assessments. A radiologically active disease was identified by Gadolinium-enhancing lesions, enlargement of an old lesion(s), or the appearance of active (i.e., new or enlarging) T2 lesions on MRI. Moreover, clinically active disease was identified in patients who had new episodes of relapse within 4 weeks after the collection of a follow-up blood sample. We also monitored the disability with EDSS.

Statistical Analyses

All statistical analyses were carried out using the Statistical Package for Social Sciences, SPSS for Windows (IBM SPSS Statistics 22, IBM Corporation, Armonk, NY, USA, 2013). Qualitative variables were described using numbers and percentages, and quantitative variables were described using medians and interquartile ranges. A non-parametric ANOVA, namely the Friedman test (at three different time points: baseline, 6 months, and 12 months), and the Wilcoxon test (at two different time points: baseline and 6 months, baseline and 12 months, 6 months and 12 months) were conducted to examine the effect of Vit-D supplementation on the levels of cytokines and other biomarkers and on the EDSS score at follow-up. The differences in cytokine ratios between the two independent groups were tested with the Mann-Whitney U test. A p value <0.05 was considered statistically significant.

Results

Demographic and Clinical Features of Cohort

All the patients were prospectively diagnosed as having relapsing-remitting MS. Among the patients, male:female ratio was 7 (43.8%):9 (56.3%). The mean and median age of our study cohort were 30.6 ± 6.3 and 31.50 (range: 20–40 years) (Q1–Q3 = 27–34.8). The demographic parameters of the cohort are represented by Table 1 . The EDSS of the cohort at recruitment was 1.4 ± 1.2.

Table 1. Demographic characteristics of the cohort Age at recruitment (mean±SD) ( N = 16) Gender, % BMI (mean±SD) ( N = 16) male ( N = 7) female ( N = 9) 30.3±7.1 years 43.8% 56.3% 27.5±5.9 The quantitative variables (age and BMI) are represented as the mean ± standard deviation, and the qualitative variable (gender) is represented as a percentage.

Demographic characteristics of the cohort

The quantitative variables (age and BMI) are represented as the mean ± standard deviation, and the qualitative variable (gender) is represented as a percentage.

Baseline Biochemical and Immunological Analysis

Baseline biochemical assessment revealed that all subjects were Vit-D deficient (16.8 [12.4–31.4] nmol/L) expressed in the median (interquartile range) ( Table 2 ). Thirteen patients had normal iPTH (5.5 [4.2–6.9] pmol/L), while 3 patients showed an elevated iPTH (mean ± SD: 12.8 ± 1.5 pmol/L) ( Table 2 ). Patients with iPTH ( N = 13) levels within the reference range were given calcium carbonate (600 mg) + Vit-D (400 units) twice a day, whereas patients with an elevated iPTH were prescribed 50,000 IU Vit-D once weekly and calcium carbonate; 600 mg + Vit-D; 400 units twice daily. In addition to Vit-D supplementation, patients were also prescribed immunotherapy. The majority of the patients ( N = 10) accepted immunotherapy, but 3 patients declined. Baseline biochemical analysis showed the presence of an overall increased level of pro-inflammatory cytokines in our subjects; specifically, serum levels of TNF-α, IFN-γ, and IL-12 were found to be significantly higher when compared to the 12-month follow-up sample ( Table 2 ).

Table 2. Changes in vitamin D (Vit-D), intact parathyroid (iPTH) hormone and cytokine levels, biochemical parameters of bone metabolism, and inflammatory markers during follow-up Variables At recruitment 6-month follow-up 12-month follow-up
p value a
p value b
p value c
p value d Vit-D, nmol/L 16.8 (12.4–31.4) 48.5 (30.6–60.6) 34.6 (28.8–60.6) 0.000 0.001 0.224 0.002 iPTH, pmol/L 6.5 (4.3–8.3) 5.6 (4.5–7.1) 6.2 (4.1–7.3) 0.280 0.683 0.394 0.427 TNF-α, pg/mL 4,061.4 (1,152.8–7,111.2) 1,991.9 (810.9–3,218.3) 894.1 (251.7–2,971.4) 0.004 0.048 0.050 0.039 INF-γ, pg/mL 15,517.8 (2,800.9–18,512.8) 10,144.8 (1,617.6–20,170.8) 3,216.4 (907.9–11,125.4) 0.116 0.650 0.055 0.041 IL-12, pg/mL 11 (3–25) 2.1 (2.1–12.4) 2.1 (2.1–6.2) 0.035 0.182 0.080 0.023 IL-4, pg/mL 2.3 (1.3–27.7) 1.3 (1.3–5.1) 1.3 (1.3–2.1) 0.327 0.139 0.314 0.055 IL-5, pg/mL 86.2 (24.1–105.1) 38.9 (7.1–128.9) 37.1 (10.4–145.6) 0.943 0.875 0.701 0.730 IL-6, pg/mL 11,050.9 (6,868.2–17,773.1) 12,709.4 (8,390.9–20,275) 9,411.5 (3,244.9–16,455.6) 0.210 0.642 0.063 0.438 IL-10, pg/mL 1,021.9 (643.4–1,661.4) 840.8 (629.6–2,001.8) 950.6 (320.6–1,765.3) 0.570 0.836 0.278 0.796 CRP, mg/dL 0.4 (0.1–0.3) 0.5 (0.14–0.4) 0.5 (0.1–0.3) 0.767 0.234 0.964 0.972 ESR, mm/h 35.4 (10.6–58.6) 34.5 (9–52.0) 27.4 (11.0–47.0) 0.331 0.734 0.799 0.204 Calcium, mmol/L 2.3 (2.3–2.4) 2.4 (2.3–2.4) 2.4 (2.3–2.4) 0.203 0.123 0.244 0.700 Corrective calcium, mmol/L 2.3 (2.3–2.3) 2.4 (2.3–2.5) 2.3 (2.3–2.4) 0.175 0.116 0.255 0.700 Albumin, gm/L 40.5 (38.5–44) 39 (37–41) 40.5 (38–44.5) 0.06 0.06 0.12 0.610 Phosphorus, mmol/L 1.2 (1.1–1.4) 1.2 (1.1–1.3) 1.21 (1.2–1.30) 0.886 0.683 0.798 0.977 Alkaline phosphate, IU/L 64.9 (47.3–82.5) 60.5 (48.0–70.0) 63.0 (51.5–74.0) 0.815 0.706 0.977 1.00 Vit-D, iPTH level, and cytokine levels and expressed in median (interquartile range).
a Friedman test to compare cytokine values of baseline, 6 months, and 12 months.
b Wilcoxon test to compare cytokine values of baseline and 6 months.
c Wilcoxon test to compare cytokine values of 6 months and 12 months.
d Wilcoxon test to compare cytokine values of baseline and 12 months.

Changes in vitamin D (Vit-D), intact parathyroid (iPTH) hormone and cytokine levels, biochemical parameters of bone metabolism, and inflammatory markers during follow-up

Vit-D, iPTH level, and cytokine levels and expressed in median (interquartile range).

a Friedman test to compare cytokine values of baseline, 6 months, and 12 months.

b Wilcoxon test to compare cytokine values of baseline and 6 months.

c Wilcoxon test to compare cytokine values of 6 months and 12 months.

d Wilcoxon test to compare cytokine values of baseline and 12 months.

Follow-Up Biochemical and Immunological Analysis

At follow-up, we found that irrespective of immunotherapy administered, Vit-D supplementation increased the Vit-D levels (Q (2) 17.4, p < 0.001) ( Table 3 ). After Vit-D supplementation, we noticed a decreasing trend in iPTH levels at the 6-month follow-up; however, it did not reach statistical significance ( Table 2 ). This finding is unusual and perhaps somewhat peculiar to MS ( Table 2 ). We observed a gradual and significant decrease in levels of the pro-inflammatory cytokines TNF-α (Q (2) 9.4, p = 0.009) and IL-12 (Q (2) 6.7, p = 0.035) ( Tables 2 , 3 ). A gradual decrease in levels of IFN-γ was also noted, but it failed to reach statistical significance. The increase in Vit-D levels and decrease in levels of the pro-inflammatory cytokines TNF-α and IL-12 are depicted in Table 2 . At follow-up, we did not find any significant differences between the levels of cytokines in the patients treated with IFN-β1α ( n = 13) and those who refused immunotherapy ( n = 3). None of the other cytokines tested, IL-4, IL-5, IL-6, and IL-10, showed any significant changes over the follow-up. Other inflammatory biomarkers, such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), did not reveal any significant change during the follow-up. Vit-D supplementation did not significantly affect biochemical parameters of bone metabolism (e.g., calcium, phosphate, and alkaline phosphatase) and is, therefore, not contributory to the changes described. Finally, there was no significant change in EDSS (Q (2) 3.4, p = 0.187) over 6 and 12 months of follow-up.

Table 3. Non-parametric ANOVA (Friedman test) comparing baseline and final follow-up (12 months) for vitamin D (Vit-D), intact parathyroid hormone (iPTH), and cytokine levels in MS patients Biomarker Q (2)
p value TNF-α 9.4 0.009 IL-12 6.7 0.035 IFN-γ 4.7 0.093 IL-6 3.1 0.210 IL-4 1.7 0.436 IL-10 1.1 0.570 IL-5 0.5 0.786 Vit-D 17.4 <0.001 iPTH 2.5 0.280 Cytokine levels are expressed in median, Q (2), the second quartile.

Non-parametric ANOVA (Friedman test) comparing baseline and final follow-up (12 months) for vitamin D (Vit-D), intact parathyroid hormone (iPTH), and cytokine levels in MS patients

Cytokine levels are expressed in median, Q (2), the second quartile.

Ratio of Pro-Inflammatory to Anti-Inflammatory Cytokines

The ratios of pro-inflammatory cytokines to anti-inflammatory cytokines provide insight into the bias or dominance of one group of cytokines over another; thus, cytokine ratios are more valuable than absolute levels of cytokines. Keeping in mind the notion that cytokine levels by themselves may not be as informative as the relative levels of pro-inflammatory to anti-inflammatory cytokines, we also calculated the ratios of median levels of cytokines.

A steady decline was observed in several ratios of pro-inflammatory to anti-inflammatory cytokines over 12 months of follow-up ( Table 4 ). For example, the TNF-α:IL-4 ratio declined significantly from 598.3 to 390.9 with 12 months of Vit-D supplementation. At the 12-month follow-up, the TNF-α:IL-5 ratio showed a significant 6-fold decrease, and the TNF-α:IL-6 ratio showed a significant 4-fold decline. Furthermore, a considerable decrease in the TNF-α:IL-10 ratio was also noted ( Table 4 ). A gradual and significant decrease was also evident in the ratios of the pro-inflammatory cytokine IL-12 to the anti-inflammatory cytokines IL-5, IL-6, and IL-10 after Vit-D supplementation ( Table 4 ). We noticed a decreasing trend in ratios involving IFN-γ, though, in most cases, the decrease in ratios did not reach statistical significance. The IFN-γ:IL-10 ratio decreased significantly from 9.8 at recruitment to 7.9 at 6 months to 5.4 at the 12-month follow-up ( p = 0.050) ( Table 5 ).

Table 4. Cytokine ratios at baseline, 6-month, and 12-month follow-up Cytokine ratios Baseline 6 months 12 months
p value a
p value b
p value c
p value d TNF-α:IL-4 598.3 (129.1–1,659.1) 918.6 (240.9–1,239.5) 390.9 (91.7–854.2) 0.273 0.959 0.386 0.203 TNF-α:IL-5 75.2 (48.8–152.4) 46.1 (17.9–98.2) 12.9 (9.1–23.1) 0.008 0.208 0.012 0.025 TNF-α:IL-6 0.3 (0.2–0.5) 0.2 (0.04–0.20) 0.11 (0.05–0.22) 0.013 0.013 0.033 0.534 TNF-α:IL-10 2.9 (2.2–5.5) 1.2 (1.1–2.9) 1.2 (0.3–1.6) 0.002 0.060 0.002 0.084 IFN-γ:IL-4 1,036.1 (463.6–4,986.2) 1,244.3 (732.3–4,474.8) 2,221.5 (432.2–10,158.2) 0.695 0.790 0.182 0.477 IFN-γ:IL-5 186.3 (106.2–266.2) 94.6 (72.2–241.2) 104.0 (16.1–398.4) 0.497 0.445 0.799 0.721 IFN-γ:IL-6 0.7 (0.4–1.7) 0.6 (0.1–0.9) 0.5 (0.2–1.5) 0.794 0.173 0.600 0.701 IFN-γ:IL-10 9.8 (4.8–16.1) 7.9 (1.9–22.9) 5.4 (1.06–9.01) 0.050 0.807 0.013 0.133 IL-12:IL-4 1.6 (0.6–2.3) 1.6 (1.6–1.8) 1.6 (1.5–1.6) 0.368 0.678 0.929 0.249 IL-12:IL-5 0.376 (0.099–0.847) 0.127 (0.059–0.541) 0.088 (0.042–0.130) 0.008 0.214 0.008 0.173 IL-12:IL-6 0.0014 (0.0004–0.0022) 0.0004 (0.0002–0.0012) 0.0004 (0.0002–0.0007) 0.035 0.110 0.033 0.424 IL-12:IL-10 0.0109 (0.0047–0.0166) 0.0043 (0.0027–0.0096) 0.0037 (0.0019–0.0082) 0.017 0.136 0.028 0.695
p values were generated using the following tests.
a Friedman test to compare baseline, 6 months, and 12 months of follow-up values.
b Wilcoxon signed rank test for baseline vs. 6 months.
c Wilcoxon signed rank test for baseline vs. 12 months.
d Wilcoxon signed rank test for 6 months vs. 12 months.

Cytokine ratios at baseline, 6-month, and 12-month follow-up

p values were generated using the following tests.

a Friedman test to compare baseline, 6 months, and 12 months of follow-up values.

b Wilcoxon signed rank test for baseline vs. 6 months.

c Wilcoxon signed rank test for baseline vs. 12 months.

d Wilcoxon signed rank test for 6 months vs. 12 months.

Table 5. Comparison of cytokine ratios according to MS relapse Cytokine ratios Relapse within first 6 months of follow-up Relapse within second 6 months of follow-up no yes (≥1)
p value no yes (≥1)
p value TNF-α:IL-4 449.8 (148.8–1,041.6) 118.8 (59.7–1,391.5) 0.353 415.8 (99.1–789.8) 365.9 (5.5–1,018.5) 0.694 TNF-α:IL-5 45.8 (10.8–101.4) 88.7 (20.6–638.0) 0.145 22.7 (12.3–69.7) 55.5 (5.0–600.0) 0.655 TNF-α:IL-6 0.11 (0.03–0.2) 0.16 (0.02–0.45) 0.575 0.07 (0.04–0.24) 0.189 (0.077–0.308) 0.454 TNF-α:IL-10 1.4 (0.4–2.5) 2.57 (1.0–4.3) 0.445 1.2 (0.6–1.6) 2.92 (0.61–11.59) 0.292 IFN-γ:IL-4 1,264.4 (521.1–3,029.1) 711.1 (492.9–3,652.4) 0.416 900.8 (179.7–4,796.2) 680.8 (192.3–3,861.2) 0.535 IFN-γ:IL-5 172.6 (47.6–435.1) 106.4 (73.4–201.3) 0.467 105.7 (26.3–307.7) 319.0 (83.3–620.6) 0.224 IFN-γ:IL-6 0.23 (0.1–1.0) 0.71 (0.2–0.8) 0.708 0.61 (0.1–1.1) 0.39 (0.2–0.7) 0.767 IFN-γ:IL-10 4.7 (2.3–9.5) 61 (1.5–11.1) 0.799 5.2 (1.1–11.8) 5.8 (1.5–8.1) 0.767 IL-12:IL-4 1.6 (1.6–1.9) 1.6 (1.5–1.9) 0.555 1.6 (0.9–1.6) 1.6 (1.0–1.6) 0.748 IL-12:IL-5 0.12 (0.05–0.39) 0.42 (0.1–0.5) 0.270 0.09 (0.02–0.2) 1.4 (0.1–9.2) 0.044 IL-12:IL-6 0.0003 (0.0002–0.0007) 0.0005 (0.0002–0.0029) 0.467 0.0003 (0.0001–0.0007) 0.0063 (0.0003–0.0064) 0.066 IL-12:IL-10 0.0052 (0.0028–0.0117) 0.0128 (0.0027–0.0413) 0.614 0.0038 (0.0020–0.0088) 0.0226 (0.0078–0.0670) 0.045
p values are generated using the Mann-Whitney U test.

Comparison of cytokine ratios according to MS relapse

p values are generated using the Mann-Whitney U test.

Relationship of the Ratio of Pro-Inflammatory to Anti-Inflammatory Cytokines with MS Relapse

We also observed some positive correlations between the number of relapses and ratios of pro-inflammatory to anti-inflammatory cytokines ( Table 5 ). Patients with higher ratios of pro-to-anti-inflammatory cytokines were more likely to have one or more relapses. However, significant relationships were found between the number of relapses and only the IL-12:IL-5 and IL-12:IL-10 ratios during the second 6-month follow-up ( Table 5 ).

Disease Severity

There was no significant change in EDSS (Q (2) 3.4, p = 0.19) over time. Moreover, there were no associations observed between cytokine ratios and radiologically active disease.

Discussion

In this investigation, the aim was to assess the impact of Vit-D supplementation on cytokine profiles in newly diagnosed, drug-naïve MS patients. Previous research has established a connection between Vit-D deficiency and autoimmune diseases [ 3 – 5 ], supported by evidence of VDRs in immune cells and Vit-D hormone production by activated dendritic cells, underscoring its immunoregulatory potential [ 3 , 4 ]. Participants in this study had very low baseline Vit-D levels. Supplementation significantly increased Vit-D levels, reduced parathyroid hormone, and decreased potent pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-12 ( Table 2 ). Over 12 months, there was a notable decline in the ratio of pro-to-anti-inflammatory cytokines, suggesting a shift away from inflammatory reactivity.

Baseline Levels of Pro-Inflammatory Cytokines

Our observation of elevated baseline serum levels of both pro-inflammatory (TNF-α, IFN-γ, IL-6) and anti-inflammatory (IL-5, IL-10) cytokines aligns with the concurrent occurrence of inflammatory and restorative processes in MS. This finding is consistent with previous research by Martins et al. [ 17 ], which reported significantly higher levels of both cytokine types in MS patients compared to controls. However, some cytokine values in our study were substantially higher than those reported by Martins et al. [ 17 ]. This discrepancy may be attributed to differences in patient characteristics (newly diagnosed vs. chronic MS) and analytical procedures used [ 17 – 19 ]. Moreover, a recent study has reported high concentrations of pro-inflammatory cytokines such as IL-12 in the serum and IL-6 and IFN-γ in the cerebrospinal fluid of MS patients [ 18 ].

MS and Pro-Inflammatory Cytokines

While the precise immune pathogenesis of MS remains unclear, IFN-γ released by Th1 lymphocytes is believed to activate astrocytes, leading to the production of inflammatory chemokines like CCL5 and MIF [ 20 ]. These chemokines promote inflammation in the brain, resulting in leukocyte infiltration and demyelination, ultimately forming sclerotic plaques [ 20 ]. Activated pro-inflammatory cytokines regulate lymphocyte movement across the blood-brain barrier, and T-cell-mediated autoimmune processes [ 20 ], influenced by genetic predisposition and environmental triggers, initiate and drive the disease [ 21 ].

Vit-D, Pro-Inflammatory Cytokines, and MS

Previous research in Kuwait has highlighted the potential role of sociocultural and environmental factors, including low sun exposure (indicative of low Vit-D levels), in the development of MS [ 21 ]. Hypovitaminosis D has been strongly associated with MS [ 22 – 24 ], and our previous studies have demonstrated a negative impact of low serum 25-hydroxy-Vit-D [25 (OH)D3] on MS status and severity, particularly in newly diagnosed, drug-naïve patients [ 25 ]. In the current study, the increased ratios of pro-to anti-inflammatory cytokine ratios reinforce the connection between Vit-D deficiency and immune dysregulation in MS [ 26 ].

Vit-D deficiency may contribute to MS pathogenesis through several mechanisms [ 3 ]. For example, it can lead to reduced intestinal calcium absorption, increased gut permeability, and subsequent translocation of endotoxins (like lipopolysaccharides) into the bloodstream [ 26 ]. LPS can stimulate the production of inflammatory cytokines within the central nervous system, particularly in the pineal gland, resulting in tissue damage [ 26 ]. Conversely, high circulating Vit-D levels are associated with a lower risk of MS [ 23 ].

Effect of Vit-D Supplementation

Our observation of decreased ratios of pro-to anti-inflammatory cytokines following Vit-D supplementation suggests a reduction in pro-inflammatory cytokine bias. This finding supports the study by Mohan et al. [ 27 ], who observed decreased IL-2 mRNA levels following Vit-D supplementation in MS patients [ 27 ]. The decreased pro-inflammatory cytokine bias is also consistent with previous findings by Mrad et al. [ 28 ], who reported an association between increased serum 25(OH)D3 levels and decreased IFN-γ production. Addition of 1,25(OH)2D to cultures of T cells from MS patients has been shown to reduce inflammatory Th17 cells and increase anti-inflammatory IL-10-producing cells [ 28 ]. Moreover, 1,25(OH) D2 and D3 have been shown to inhibit the proliferation of both CD4 + T cells and MBP-specific T cells [ 29 ]. As T cells express alpha1-hydroxylase activities, they can metabolize 25(OH) D3 into biologically active 1,25(OH) 2 D3. Activated Vit-D was shown to enhance the development of anti-inflammatory IL-10-producing cells and reduce the number of pro-inflammatory IL-6-secreting cells [ 30 ]. Vit-D supplementation in our patients increased the level of activated Vit-D, which could have contributed to the reduced pro-inflammatory cytokine bias observed in our study. Moreover, Vit-D supplementation could have brought about a decrease in relapse rates by lowering the inflammatory cytokine bias.

We infer that the increased levels of Vit-D and the decreased levels of pro-inflammatory cytokines following Vit-D supplementation suggest that patients with MS would benefit from increasing their intake of Vit-D. We found a significant decline in pro-inflammatory cytokines irrespective of immunotherapy, but the role of immunotherapy in this decline cannot be ruled out. The strengths of this study are the inclusion of incident MS cases, which is usually rare in the literature, as well as the measurement of several cytokines rather than just one or two and the calculation of cytokine ratios as a measure of cytokine dominance. We suggest that examining the ratios of pro-to anti-inflammatory cytokines is an interesting and important way of assessing inflammatory bias or dominance; this approach has not been described in previous reports on the influence of Vit-D on cytokine patterns in MS patients. Though the sample size is adequate to represent the target population of this study, it is important to conduct similar studies with larger sample sizes to better understand the beneficial roles of Vit-D supplementation in MS.

Conclusions

High levels of pro-inflammatory cytokines and low levels of Vit-D in drug-naïve patients strongly support the contention that hypovitaminosis D may play an immune-patho and genetic role in the development of MS in our study cohort. Moreover, Vit-D supplementation possibly plays a protective role in the prognosis of MS through an immune-pathologic pathway and is of therapeutic importance.

Statement of Ethics

This study has been carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Written informed consent was taken from all the patients included in this study. This study protocol was reviewed and approved by the Ethics Committees of the Health Sciences Centre, Kuwait University, Grant No. MM 03/09.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by the funding by the Ethics Committees of the Health Sciences Centre, Kuwait University, Grant No. MM 03/09.

Author Contributions

Suhail Al-Shammri contributed to visualization, conceptualization, resources, funding acquisition, recruitment of patients, writing – review and editing. Arpita Chattopadhyay contributed to software, validation, formal analysis, investigation, data curation, writing – original draft preparation, and writing – review and editing. Raj Raghupathy contributed conceptualization, supervision, methodology, resources, and writing – review and editing.

Funding Statement

This work was supported by the funding by the Ethics Committees of the Health Sciences Centre, Kuwait University, Grant No. MM 03/09.

Data Availability Statement

The data that support the findings of this study are available on request from Suhail Al-Shammri or the corresponding author.

References

Associated Data

Data Availability Statement

The data that support the findings of this study are available on request from Suhail Al-Shammri or the corresponding author.